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Yeast Nitrogen Base Minus Amino Acids (YNB-AA) Recipe

  • For 1 L Batch:
    • 1.7g Yeast Nitrogen Base (stored in cold room)
    • 5g (NH4)2SO4 (Ammonium Sulfate, stored in chemical stock cabinet, EMD

168355)

  • Dissolve in 800mL Np H2O for YNB-AA stock
  • Adjust pH value to 5.8 using NaOH (starting May 2011) -Yen-Hsiang

Autoclave (store at RT for media stocks)

To use as working media:

  • After autoclaving add:
    • 100mL 10X (20%) Dextrose (BD 215530)
    • 100mL 10X Drop Out stock (10X DO stock recipe here: [[1]])
    • 1 L Total Volume

Yeast Nitrogen Base Minus Amino Acids Non-Inducing, Non-Repressing (YNB-AA NINR) Recipe

  • For 1 L Batch:
    • 1.7g Yeast Nitrogen Base
    • 5g NH3SO4
  • Dissolve in 700mL Np H2O for YNB-AA NINR stock
  • Adjust pH value to 5.8 using NaOH (starting May 2011) -Yen-Hsiang
  • Autoclave (store at RT for media stocks)

To use as working media:

  • After autoclaving add:
    • 100mL 10X (20%) Raffinose (BD 217410)
    • 100mL 10X (10%) Sucrose (Sigma S3929)
    • 100mL 10X Drop Out stock
    • 1 L Total Volume

Yeast Peptone (YP) Media Recipe

  • For 1 L Batch:
    • 10g Yeast Extract [Bacto™ REF#212750] (stored in chemical stock cabinet)
    • 20g Peptone [Bacto™ REF#211677] (stored in chemical stock cabinet)
    • 80 mg of Adenine hemisulfate (Sigma Chemical Co. Ltd., cat. no. A-3159) (stored in chemical stock cabinet)
    • Dissolve in 900mL Np H2O for YP stock

Autoclave (store at RT for media stocks) To use as working media: After autoclaving add:

    • 100mL 10X (20%) Dextrose
    • 1 L Total Volume YPD (Yeast Peptone Dextrose)

To pour any of these media stocks as plates:

    • Add 20g agar per 1L final media volume before autoclaving
    • See here [[2]]

For yeast KanMX (G418) selection:

  • Cannot use ammonium sulfate as primary nitrogen source
  • Add G418 to YP or to YNB with 1 g/L MSG (Sigma G1626) instead of ammounium sulfate
  • Aqueous solutions stored frozen at -20 to -70 °C should be stable for approximately six months.
  • Powder G418 sulfate (Geneticin) is stored in chemical room.
  • Add 400mg/L for selection (in plates)
  • Add 200mg/L for maintenance (in liquid culture media)

For yeast counterselection:

  • Add 1 g/l of 5-FOA to YNB-SC solid media to counterselect for URA3
    • While adding 20% dextrose and 10x SC AA mix, have 5-FOA dissolved in DMSO and add before stirring and pouring out agar (for a typical single sleeve 500ml media batch, this can be 500mg of 5-FOA in 5ml DMSO)
  • Add 1.3 g/l of 5-fluorocytosine (5-FC) to YNB-SC solid media to counterselect for FCY1
  • For alpha-AAD/LYS2,5 counterselection replace nitrogen source with 2g/L (0.2%) D,L-alpha-aminoadipate (Bachem, F-1185)
    • dissolve 1 gram alpha-AAD in 20ml of water and pH with KOH to pH 6.0 (otherwise insoluble), filter sterilize and add to YNB with special AA mix
    • two recipes have been shown to work for this counterselection

-Michael S. Siddiqui 21:53, 4 January 2011 (EST)* 01/04/2011 (aided by Andrew Babiskin)